Infection with a subset of high-risk human papillomaviruses (HPV) is the major risk factor for cervical cancer. The HPV-16 E6 and E7 genes are selectively retained and expressed in most cervical carcinomas, and persistent infection with HPV and immortalization of cervical cells are important early events in cervical carcinogenesis. Although infection with HPV is required for malignant progression, it is not sufficient. Most high-risk HPV infections are recognized and eliminated by the host's immune response; only a small subset progress to invasive cervical cancer. We are interested in two important questions: (1) what is unique about infections that progress and (2) what are the critical cofactors that promote cervical carcinogenesis in HPV-infected cells. Although HPV infections occur throughout the cervix, approximately 90% of cervical cancers occur within a small anatomic region known as the transformation zone. The transformation zone consists of a narrow band of metaplastic squamous epithelium between the stratified squamous epithelium of the ectocervix and the columnar secretory epithelium of the endocervix. Almost all cervical cancers originate within the transformation zone, but it is unclear why HPV-infected cells from this region have such a high probability of progression. We hypothesize that cells from the transformation zone are more susceptible to immortalization by HPV-16 than cells from the surrounding ecto and endocervix. We also predict that HPV-16-immortalized cells derived from transformation zone are most susceptible to proliferation and dysplastic differentiation in response to two cofactors that promote cervical carcinogenesis, chronic inflammation and estrogen. To examine these questions, human epithelial cells will be cultured from the transformation zone, ectocervix, and endocervix. The cultured cells will be compared for susceptibility to immortalization by HPV-16, and potential mechanisms that contribute to differences in immortalization frequency will be examined. HPV-immortalized epithelial cells from each anatomic region will also be assessed for their response to two cofactors that contribute to cervical cancer, estrogen and the proinflammatory cytokine, tumor necrosis factor-alpha (TNF-). Because most cervical cancers arise from the transformation zone, it is logical to focus on how these cells interact with HPV. If our experiments are successful, our results may help to identify important signal pathways or biomarkers that are unique to transformation zone cells and that can be targeted for improved screening, prevention or therapy.